Induction of Differential T-Cell Epitope by Plain- and Liposome

Liposomal conjugates with plain OVA, Alexa−OVA, or DQ−OVA were prepared essentially in the same way as described previously (15) via glutaraldehyd...
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Bioconjugate Chem. 2006, 17, 899−904

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Induction of Differential T-Cell Epitope by Plain- and Liposome-Coupled Antigen Maiko Taneichi,† Yuriko Tanaka,† Michiyuki Kasai,† Masahito Mori,‡ Mitsuhiro Nishida,‡ Hiroyuki Yamamura,‡ Junichiro Mizuguchi,§ and Tetsuya Uchida†,* Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo 208-0011, Japan, Drug Delivery System Development Division, Nippon Oil and Fat Corporation, Tokyo 150-6019, Japan, and Department of Immunology, Tokyo Medical University, Tokyo 160-8402, Japan. Received January 31, 2006; Revised Manuscript Received April 30, 2006

The T-cell receptors of CD4+ T lymphocytes recognize immunogenic peptide sequences bound within the groove of MHC class II molecules, and the peptides that bind to these molecules are known to share common structural motifs. For example, OVA323-339, an I-Ad-binding peptide, involves a motif of the I-Ad peptide-binding groove. In the present study, OVA peptides of up to 26-mer were sequentially synthesized and screened, and two additional I-Ad binding OVA peptides, OVA20-43 and OVA264-286, were found to stimulate CD4+ T cells of OVA-immune BALB/c mice. OVA20-43 involved structural motifs of the I-Ad peptide-binding groove, while OVA264-286 did not. The ability of these three I-Ad binding OVA peptides to induce antigen-specific cytokine production was compared among CD4+ T cells of mice immunized either with alum-adsorbed OVA (OVA-alum) or OVA chemically coupled to the surface of liposome (OVA-liposome). CD4+ T cells of mice immunized with OVAalum produced more cytokines when stimulated with OVA264-286 than with OVA323-339, while CD4+ T cells of mice immunized with OVA-liposome conjugates produced more cytokines when stimulated with OVA323-339 than with OVA264-286. OVA20-43 induced production of comparable levels of cytokines in mice immunized either with OVA-alum or OVA-liposome. Confocal laser scanning microscopic analysis demonstrated that chemically coupled OVA and liposomes were colocalized in APCs until OVA received processing. Three-dimensional structural analysis demonstrated that both OVA264-286 and OVA323-339 were present on the surface of OVA, but OVA20-43 was not. These results suggested that the chemical coupling of OVA to liposome affected antigen processing in APCs and thus resulted in the induction of differential T-cell epitopes as compared with those induced by plain OVA.

INTRODUCTION In general, T-cell receptors (TCRs) of CD4+ T cells do not recognize native protein antigens, but do recognize antigenic peptides displayed in association with major histocompatibility complex (MHC) class II molecules by antigen-presenting cells (APCs). TCRs occur as either of two distinct heterodimers, Rβ or γδ. The Rβ TCR heterodimer-expressing cells predominate in most lymphoid compartments (90% to 95%) of humans and mice and are responsible for the classical helper or cytotoxic T cell responses. In most cases, the Rβ TCR ligand is a peptide antigen bound to the class I and class II MHC molecule (1). Class I and II molecules encoded by genes within the MHC play a central role in regulating immune responses through their ability to bind and display small peptides derived from foreign antigens. Grey and co-workers identified ovalbumin (OVA) peptide 323-339 as being immunodominant in H-2d mice (2) and showed that this peptide binds to I-Ad, but not I-Ed, the latter of which is an Ia molecule that will not present this peptide (3). Ever since OVA323-339 was identified, it has been used extensively to study the nature of class II MHC-peptide binding and T-cell activation (4-9). However, this peptide is responsible for 25-35% of the T-cell responses in BALB/c mice immunized with whole ovalbumin (2), suggesting the presence of other I-Ad * To whom correspondence should be addressed. Tel: +81-42-5610771; Fax: +81-42-562-7892. E-mail: [email protected]. † National Institute of Infectious Diseases. ‡ Nippon Oil and Fat Corporation. § Tokyo Medical University.

binding OVA peptides with T-cell stimulatory activity while, at present, no other I-Ad binding OVA peptides have been reported. We previously reported that OVA chemically coupled to the surface of liposomes via glutaraldehyde (OVA-liposome conjugates) induced IgE-selective unresponsiveness in mice (10). The IgE-selective unresponsiveness was induced by antigenliposome conjugates, regardless of the coupling procedure of antigen and liposomes (11), using liposomes of different lipid formulations (12), or using different antigens such as tetanus toxin (13) or Shiga-like toxin (14). Thus, antigen-liposome conjugates are expected to be applicable as part of a novel protocol for the development of vaccines that would induce minimal IgE synthesis. In the present study, sequentially synthesized OVA peptides were screened for the ability to stimulate OVA-specific CD4+ T cells of BALB/c (H-2d) mice and two additional I-Ad binding OVA peptides, OVA20-43 and OVA264-286, were found. The ability of these three OVA peptides to stimulate CD4+ T cells of OVA-immune BALB/c mice was compared among the CD4+ T cells of mice immunized either with OVA-alum or with OVA-liposome conjugates. The aim of this study was to investigate the effect of antigen modification on the in vivo induction of T cell epitopes.

EXPERIMENTAL PROCEDURES Mice. BALB/c mice (female, 8 weeks of age) were purchased from Charles River, Kanagawa, Japan. The mice were maintained in sterile cages under specific pathogen-free conditions at the Division of Experimental Animals Research (National Institute of Infectious Diseases, Tokyo, Japan).

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Antigens. Ovalbumin (OVA, Grade VII) was purchased from Sigma (St. Louis, MO). For analysis of the processing of liposome-coupled OVA by macrophages, DQ-OVA, which exhibits green fluorescence upon proteolytic degradation, was purchased from Molecular Probes, Inc. (Eugene, OR). OVA Peptides. OVA peptides consisting of 20-26 amino residues were sequentially synthesized, and the ability of each peptide to induce cytokine production by CD4+ T cells of BALB/c mice immunized with OVA was investigated. All OVA peptides used in the present study were synthesized and supplied by Operon Biotechnologies, K. K. (Tokyo, Japan). Precursor peptide material attached to polystyrene resin was synthesized by an automated peptide synthesizer (model 433A; Applied Biosystems, Foster City, CA) using Fmoc (9-fluorenylmethoxycarbonyl) chemistry. Then, the resins on the peptide C-terminus and the building blocks on the peptide side chain were removed by trifluoroacetic acid. The purification of the peptide was performed by high-performance liquid chromatography (HPLC) using an automated system (HPLC-10A; Shimazu Co., Kyoto, Japan) with a 35-55% acetonitrile gradient in the acidic aqueous phase. As a result, the highly purified material was obtained with a >95% area ratio on the HPLC elution chart and was determined by mass spectroscopic analysis with the matrixassisted laser desorption/ionization time-of-flight method using Applied Biosystems Voyager DE. The peptide solution was lyophilized and used for the subsequent experiments. Fluorescence Labeling of OVA. OVA were labeled with fluorescence using an Alexa Fluor 488 protein labeling kit (Molecular Probes, Inc.) following the manufacturer’s protocol. The estimated F:P ratio of Alexa Fluor 488-OVA conjugate was 1.4:1. Chemicals. All phospholipids were obtained from NOF Co., Tokyo, Japan. Reagent grades of cholesterol were purchased from Wako Pure Chemical Industries, Osaka, Japan. Liposomes. The liposomes used in the present study consisted of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidyl ethanolamine, cholesterol, and dimyristoyl phosphatidyl glycerol in 4:3:7:2 molar ratios. The crude liposome solution was passed through a membrane filter (Nucleopore polycarbonate filter, Coster, Cambridge, MA) with a pore size of 0.2 µm. Red-Labeled liposome. Red-labeled liposomes were prepared by adding 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamineN-(lissamine rhodamine B sulfonyl) (Product Number: 810157, Avanti Polar Lipids Inc, Alabaster, AL) into the above lipid constituent of liposomes. Coupling of OVA to Liposomes. Liposomal conjugates with plain OVA, Alexa-OVA, or DQ-OVA were prepared essentially in the same way as described previously (15) via glutaraldehyde. Briefly, to a mixture of 90 mg of liposomes and 6 mg of OVA in 2.5 mL of phosphate buffer (pH 7.2) was added 0.5 mL drops of 2.5% glutaraldehyde solution. The mixture was stirred gently for 1 h at 37 °C, and then 0.5 mL of 3 M glycine-NaOH (pH 7.2) was added to block excess aldehyde groups. This was followed by incubation overnight at 4 °C. The liposome-coupled OVA and uncoupled OVA in the resulting solution were separated using CL-4B column chromatography (Pharmacia Fine Chemical Co., Upsala, Sweden). The amount of lipid in the liposomal fraction was measured using a Phospholipid-Test-Wako phospholipid content assay kit (Wako Pure Chemical Industries). The OVA-liposome solution was adjusted to 10 mg lipid/mL in RPMI-1640, sterile-filtered using a Millex-HA syringe filter unit (0.45 µm, Millipore Corp., Bedford, MA), and kept at 4 °C until use. Immunization. The mice were immunized intraperitoneally (ip) with 200 µL of 10 µg of OVA adsorbed with 100 µg of

Taneichi et al.

alum (Alhydrogel, Superfos Biosector, Vedbaek, Denmark) in PBS or with 200 µL of OVA-liposome conjugates, at 0 and 4 weeks. Preparation of Splenic Adherent Cells. Splenic adherent cells (SACs) were used as antigen-presenting cells in the cultures of the CD4+ T cells and OVA peptides. SACs were obtained from naive BALB/c mice as follows. Spleen cell suspensions were prepared in RPMI-1640 containing 10% fetal calf serum (FCS). Cells (5 × 107) in 5 mL of medium containing 10% FCS were plated into 50-mm plastic tissue culture dishes (No. 3002; Becton-Dickinson Labware, Franklin Lakes, NJ) and incubated at 37 °C in a humidified 5% CO2 atmosphere for 2 h. After culture, nonadherent cells were removed by three vigorous washings in warm media, and adherent cells were then harvested with a cell scraper. Preparation of CD4+ T Cells. CD4+ T-cell purification from spleen cells of mice immunized with OVA-alum was performed with the MACS magnetic cell sorter system (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s protocol and using anti-CD4 antibody-coated microbeads (No. 492-01; Miltenyi Biotec). CD4+ T cells were suspended in RPMI-1640 containing 10% FCS at a cell density of 2 × 106/mL. In the present study, OVA peptides at a final concentration of 10 µM with 1 and 5 d culture periods were employed for the production of the Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-5) cytokines, respectively. The CD4+ T-cell suspension was plated at 250 µL per well onto 48-well culture plates (No. 3047; Becton-Dickinson Labware), and 500 µL of 20 µM OVA peptide solution and 250 µL of 8 × 105/mL SAC in the same medium were added to the plates. After incubation in a CO2 incubator, the culture supernatants were collected and assayed to determine the concentrations of cytokines. Cytokine Assays. IL-5 and IFN-γ in the culture supernatant were measured using the Biotrak mouse ELISA system (Amersham International, Buckinghamshire, UK). All test samples were assayed in duplicate, and the standard error in each test was always less than 5% of the mean value. Cloned Macrophage Hybridoma. Macrophage hybridoma clone No. 39, obtained from the fusion of splenic adherent cells from CKB mice and P388D1 (16), was maintained in RPMI1640 (Gibco Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated FCS (Hyclone Laboratories, Logan, UT), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (Gibco Laboratories) in a 75 cm2 flask (No. 3111; Becton Dickinson Labware, Franklin Lakes, NJ). Flow Cytometry. To investigate the processing of OVA coupled to liposome by macrophages, #39 macrophage clone was incubated for 15 to 60 min at 37 °C in the presence of DQ-OVA-liposome conjugates that contained a final concentration of 4 µg/mL OVA. After incubation, the cells were washed with ice-cold PBS and then were analyzed on a FACS Calibur flow cytometer (BD Biosciences, Mountain View, CA). The histograms of fluorescence distribution were plotted as the number of cells versus fluorescence intensity on a logarithmic scale. Confocal Laser Scanning Microscopy. To investigate the processing of liposome-coupled OVA by macrophages, macrophage clone No. 39 was cultured for 18 h at 37°C on 8-hole heavy Teflon-coated slides (Bokusui Brown, New York, NY) and then incubated with DQ-OVA-liposome conjugates for 2 h at 37 °C. The slides were washed with MEM and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After fixation, they were incubated for 10 min in 0.1 M glycine-HCl, pH 7.0, to block the remaining ardehyde residue. They were then washed two times in PBS. After washing, the slides were sealed with PBS/glycerin (PBS:glycerin ) 1:9). They were then analyzed under a confocal laser scanning

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Table 1. OVA Peptides Investigated in the Present Study OVA peptides

amino acid sequence

1-20 20-43 53-78 61-85 164-181 190-207 220-245 264-286 302-327 323-339

GSIGAASMEFCFDVFKELKV VHHANENIFYCPIAIMSALAMVYL INKVVRFDKLPGFGDSIEAQCGTSVN KLPGFGDSIEAQCGTSVNVHSSLRD SSVDSQTAMVLVNAIVFK DEDTQAMPFRVTEQESKP ASMASEKMKILELPFASGTMSMLVLL LTEWTSSNVMEERKIKVYLPRMK ITDVFSSSANLSGISSAESLKISQAV ISQAVHAAHAEINEAGR

Table 2. Induction of Cytokine Production by OVA Peptidesa OVA peptides

IL-2

IL-4

IL-5

IFN-γ

1-20 20-43 53-78 61-85 164-181 190-207 220-245 264-286 302-327 323-339 OVA

ND 278.3 ( 53.5 ND ND ND ND ND 511.7 ( 85.0 ND 215.6 ( 35.5 856.7 ( 60.8

ND 221.2 ( 78.1 ND ND ND ND ND 140.3 ( 28.3 ND 27.8 ( 15.1 78.0 ( 30.1

ND 1201.5 ( 58.3 ND ND ND ND ND 1073.4 ( 156.3 ND 560.1 ( 51.1 1133.4 ( 39.1

ND 1275.5 ( 507.9 ND ND ND ND ND 1321.6 ( 260.7 ND 302.6 ( 72.6 2457.0 ( 667.3

a Each peptide was assessed for its ability to induce cytokine production by CD4 T cells of mice immunized with OVA-alum. CD4 T cells were cultured in the presence of OVA peptides as described in Experimental Procedures. Data represent mean cytokine concentration (pg/mL) and SE of triplicate cultures. ND, not detected.

microscope system, LSM410 (Carl Zeiss Co., Germany). Internalization of Alexa-OVA-red liposome conjugates in the macrophages was investigated in essentially the same manner as above. Analysis of Three-Dimensional Structure of OVA. The three-dimensional structure of OVA reported by Stein et al. (17, 18) was obtained from DBGET database links (http://www.genome.jp/dbget/), and the locations of the OVA peptides were displayed using ProteinAdviser (Fujitsu Kyusyu System Engineering Ltd., Fukuoka, Japan).

RESULTS OVA Peptides Capable of Inducing Cytokine Production by OVA-Specific CD4+ T Cells of BALB/c Mice. A series of sequentially synthesized OVA peptides shown in Table 1 were screened for the ability to induce cytokine production by CD4+ T cells of BALB/c mice immunized with OVA-alum. Since, under the culture conditions described in Materials and Methods, no cytokine production was observed in the absence of OVA, nor by the use of CD4+ T cells of naive BALB/c mice, the data shown in Table 2 were considered to represent antigenspecific cytokine production. In addition to well-known OVA323-339, two OVA peptides, OVA20-43 and OVA264-286, were found to induce significant production of all cytokines tested. The levels of cytokines induced by OVA20-43 and OVA264-286 were even higher than those induced by OVA323-339 in all cytokines tested. OVA20-43 involved the core motif of the I-Ad peptide-binding groove, OVA31-36, but OVA264-286 did not. Cytokine Production by OVA-Specific CD4+ T Cells Of Mice Immunized with OVA-Alum or OVA-Liposome, after Stimulation with OVA Peptides. The next attempt was performed using newly found OVA peptides, OVA20-43 and OVA264-286, and a well-known OVA peptide, OVA323-339. Splenic CD4+ T cells were obtained from mice immunized either with OVA-liposome or OVA-alum and were cultured with OVA20-43, OVA264-286, or OVA323-339 in the presence of

Figure 1. Cytokine production by CD4+ T cells of mice immunized with OVA-liposome or OVA-alum, after in vitro stimulation with OVA peptides. CD4+ T cells were taken from OVA-immune mice and cultured with OVA peptides as described in Materials and Methods. Data represent mean cytokine concentration (pg/mL) and SE of triplicate culture. Data are representative of three independent experiments.

APCs. As shown in Figure 1, the levels of cytokines produced by CD+ T cells of mice immunized with OVA-liposome were significantly (p < 0.01) higher when the T cells were cultured either with OVA20-43 or OVA323-339 than when they were cultured with OVA264-286. On the other hand, in the culture of the CD4+ T cells of mice immunized with OVA-alum, OVA20-43 and OVA264-286 induced significantly higher levels of IL-5 and IFN-γ as compared to those induced by OVA323-339, consistent with the results shown in Table 1. Similar results were obtained for the other cytokines, IL-2, IL-4, and IL-10 (data not shown). Duration of Processing of Liposome-Coupled OVA in APCs. DQ-OVA coupled to liposome was cultured for 15 to 60 min with macrophage clone No. 39, and FACS analysis was performed. Figure 2a shows the fluorescence intensity of macrophages. A maximal processing of OVA was observed at 60 min. A consistent result was observed by confocal microscopic analysis (Figure 2b); the green fluorescence in the macrophages increased gradually within 60 min, suggesting that liposome-coupled OVA received processing significantly within 60 min after onset of the culture. Internalization of Alexa-OVA-Red Liposome Conjugates in Macrophages. Alexa-OVA-red liposome conjugates, which appear yellow due to association with the green in Alexa-OVA and the red in the liposome, were cultured with macrophage clone No. 39 for 15 to 60 min, and confocal

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Figure 2. Digestion of liposome-coupled OVA by macrophages. DQOVA coupled to the surface of liposomes were added to the macrophage culture. Zero to 60 min after the onset of the culture, the macrophages were recovered and analyzed using flow cytometry (a) and confocal laser scanning microscopy (b). Figure 4. Location of OVA peptides in the three-dimensional structure of OVA. The OVA peptides in the three-dimensional structure of OVA are indicated as yellow strings.

Figure 3. Association of OVA and liposomes in macrophages. Conjugates of Alexa488-labeled OVA and red-labeled liposomes were added to the macrophage culture. Zero to 60 min after the onset of the culture, the macrophages were recovered and analyzed using confocal laser scanning microscopy. The yellow spots observed in the macrophages are the result of the association with the Alexa488 (green) and red-labeled liposomes.

scanning microscopic analysis was performed. As shown in Figure 3, yellow spots were observed up to 60 min, suggesting that the OVA and liposomes were associated until liposomecoupled OVA received processing (Figure 2). OVA Peptides in Three-Dimensional Structure of OVA. The locations of the three OVA peptides, OVA20-43, OVA264-286, and OVA323-339, in the three-dimensional structure of OVA were analyzed. As shown in Figure 4, both OVA264-286 and OVA323-339 located on the surface of OVA in the three-dimensional structure, while OVA20-43 was situated inside the threedimensional structure of OVA.

DISCUSSION In the present study, OVA20-43 and OVA264-286 were newly found among sequentially synthesized OVA peptides to stimulate CD4+ T cells of OVA-immune BALB/c mice for cytokine production. A length of six residues has been proposed for core motifs of the I-Ad peptide-binding groove, with the first, third, and fourth residues being hydrophobic and the sixth generally being alanine or serine (5, 19). OVA contains at least eight different epitopes with the I-Ad-binding motif, including OVA327-332 which is involved in OVA323-339. OVA20-43 involved core motifs of the I-Ad peptide binding groove, OVA31-36 and OVA32-37, while OVA264-286 did not. In addition, although OVA1-20, OVA53-78, OVA220-245, and OVA302-327

involved the core motifs OVA1-6, OVA63-68, OVA230-235, and OVA312-317, respectively, none of them induced OVA-specific cytokine production by CD4+ T cells of OVA-immune BALB/c mice (Table 2). These results suggest that the involvement of structural motifs of the MHC class II peptide binding groove in an antigenic peptide is not an essential condition for the activation of antigen-specific CD4+ T cells. Also, regarding class I, although OVA contains six potential epitopes with the H-2Kbbinding motif (20), the CTL response is dominated by just one, OVA257-264 (21-23). Thus, immunodominance would be directed by more than just the presence of an appropriate class I- or class II-binding motif. The Rβ TCR comprises disulfide-linked R and β chains, each of which has a membrane-distal variable (VR or Vβ) and membrane-proximal constant (CR and Cβ) immunoglobulin superfamily (IgSF) domain, transmembrane regions, and short cytoplasmic segments (24). The peptide-MHC binding site is formed primarily from three complementary-determining regions (CDRs) or loops contributed by each VR and Vβ domain. TCRs dock to the peptide-MHC in a topologically constrained manner, i.e., with the VR domain of the TCR positioned over the N-terminal half of the peptide and the Vβ domain over the C-terminus (25, 26). Thus, the shape of the TCR and/or peptideMHC binding surfaces might limit the number of docking orientations (24). Therefore, the processing of both OVA20-43 and OVA264-286 in APC to produce the “essential” length of the I-Ad-binding peptides sequence would be indispensable for the presentation of those peptides to CD4+ T cells via the interaction between the peptide-MHC and TCRs. Each Ia is considered to possess a single peptide binding site, based on the observation that unrelated peptides restricted by the same Ia molecule are capable of inhibiting the binding of one another to that particular Ia (27). This in turn supports the concept that a single site on Ia is capable of recognizing a structural motif common to many antigens (5). However, in the

T-Cell Epitope Induced by Plain- and Liposome-Coupled Antigen

present study, among two newly found I-Ad-binding OVA peptides, although each were found capable of stimulating antigen-specific CD4+ T cells, one was demonstrated to involve a structural motif of the I-Ad peptide-binding groove but the other was not, suggesting the existence of another motif of the I-Ad peptide-binding groove. OVA323-339 induced a significantly higher level of cytokine production by CD4+ T cells of mice immunized with OVAliposome than that induced by OVA264-286. However, in contrast, OVA323-339 induced a significantly lower level of cytokine production by CD4+ T cells of mice immunized with OVAalum than that induced by OVA264-286. These results suggest that the modification of OVA by the chemical coupling of OVA with liposomes may interrupt or alter antigen processing in APCs and result in the presentation of a differential set of peptides to T cells. In fact, the results of the present study demonstrated that the OVA and liposomes were associated until liposome-coupled OVA was processed (Figures 2 and 3). In the previous study (13), antigen-liposome conjugates were shown to be stable for at least 6 months if stored at 4 °C, and, if tetanus toxoid was coupled to liposomes and lyophilized, their efficacy was maintained for 6 months at 37 °C. The fact that both OVA264-286 and OVA323-339 were present on the surface of the three-dimensional structure of OVA made it reasonable to consider the effect of antigen-modification on the antigen processing in APCs. On the other hand, OVA20-43, which induced a comparable level of cytokine production in both OVA-alum- and OVA-liposome-immune mice, was present inside the structure of OVA and likely not affected by the antigen-modification. It is unlikely that the antigen-modification by the chemical coupling of antigen to liposomes detracts from the efficacy of vaccines when liposome-coupled antigen is applied to the vaccination protocol, since liposome-coupled toxins successfully induced protection against the tetanus toxin (13) or Shiga-like toxin (14, 28, 29) in mice (13, 14) and also in monkeys (28). Nor it is unlikely that the difference between the two OVA peptides, OVA264-286 and OVA323-339, in the ability to induce cytokine production by CD4+ T cells is reflected by the difference of immunodominance (i.e., affinity to bind I-Ad) in these two OVA peptides, since these peptides yielded opposite results in the stimulation of OVA-specific cytokine production by CD4+ T cells of mice immunized by two different ways of immunization, i.e., OVA-alum and OVA-liposome. Thus, the results in the present study suggested that the modification of antigen affects antigen processing in APCs and results in the induction of differential T-cell epitopes as compared with those induced by plain antigen.

ACKNOWLEDGMENT This work was supported in part by a grant from The Japan Health Sciences Foundation (Research on Health Sciences focusing on Drug Innovation).

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